1. Field of the Invention
The present invention relates generally to the field of spectrophotometers and more particularly to the determination of stray light within such instruments and the compensation of sample measurements for stray light.
2. Description of the Prior Art
Spectrophotometers can be described as instruments that measure the relative amount of radiant energy absorbed or transmitted by a sample for one or more radiation wavelengths. Such instruments generally include a continuum radiation source, that is, one which generates radiant energy over a relatively broad band of wavelengths. A monochromator receives the radiation from the source and isolates an output beam comprising radiation having wavelengths substantially within a relatively narrow wavelength band.
The monochromator output beam is directed to a detector which produces an electrical output having a value related primarily to the radiant power received by the detector. Such detector output may be decreased by an amount equal to the detector output when radiation is blocked from the detector, generally called "dark current" or "dark signal". The resulting net output is called "detected radiant power" (DRP) and is the portion of the detector output generated only in response to incident radiant power.
The beam path between the monochromator and the detector is accessible to the user of the instrument so that sample or reference materials can be placed into the beam. Usually, the relative transmittance or absorbance of a sample with respect to a reference material is measured. For example, for given radiation wavelengths within the narrow wavelength band, the reference material is placed into the beam between the monochromator and the detector and the resulting DRP is measured. The reference material is removed and, with the sample in its place, a second DRP is measured. The sample transmittance is then expressed as a ratio of the second DRP with respect to the first DRP. Absorbance is related to transmittance by the conversion expression A=-log T, where A is absorbance and T is transmittance. It will be recognized that although various examples and discussions included herein are presented in terms of transmittance, such examples and discussions are equally applicable to the measurement of absorbance since absorbance and transmittance are related terms for the same phenomenon.
The previously referred to monochromator wavelength band may be largely defined by two parameters, half bandwidth and central wavelength. The half bandwidth is generally identified as the difference between two wavelengths at which the DRP of the narrow wavelength band is one-half the maximum DRP in the band. The half bandwidth is usually determined by the width of monochromator entrance and exit slits through which the continuum radiation and the output beam pass, respectively. The interval or width of the narrow wavelength band is often considered to be twice the interval or width of the half bandwidth.
The central wavelength is the wavelength at the midpoint between the two wavelengths which define the half bandwidth. The central wavelength is adjusted by means of a mechanism that positions a prism or grating disperser within the monochromator. For manually adjusted spectrophotometers, the adjustment mechanism often includes a dial calibrated with respect to the central wavelength and the central wavelength is often referred to in the art as "monochromator dial setting" or simply "dial setting." More recently and particularly with microprocessor-based spectrophotometers, the adjustment mechanism is responsive and calibrated with respect to digital signals from the microprocessor with the digital signals controlling the central wavelength. Hence, where the term "dial setting" is used herein, it is to be understood as the central wavelength adjustment, regardless of the means thereof.
Ideally, the monochromator should pass only radiation having wavelengths within the narrow wavelength band, that is, the monochromator output beam should be free of radiation with wavelengths outside of an interval twice the width of the half bandwidth and centered at the central wavelength. However, such ideal monochromators do not exist. In addition to radiation with wavelengths within such an interval, which has been called "primary radiation," the monochromator output beam also includes radiation at wavelengths outside the interval of primary radiation. Such radiation has been referred to in the art as "stray light" and is often a result of light scattering within the monochromator.
While "stray light" is used herein as just described for the purpose of discussion of the prior art, it will be recognized by those skilled in the art that the term "stray light" has not been clearly defined or limited in use in the prior art. The term has been used variously to denote the overall problem of stray light in spectrophotometers, or a measured quantity of stray light, usually with unknown units, or dimensionless ratios such as stray light ratio and stray radiant energy. It will also be noted that light is synonymous with "radiation" and that "radiation" as used herein includes electromagnetic radiation throughout the ultraviolet, visible and infrared wavelength regions. The term "stray light" will be defined more precisely hereinbelow in the detailed description of the present invention.
Stray light is usually of interest in two contexts. First, it is a general practice of spectrophotometer instrument manufacturers to measure stray light for a particular spectrophotometer and to publish the measurement as an instrument performance specification. Periodically, a spectrophotometer should be retested to determine whether the spectrophotometer still meets the specification. A failure of the instrument to do so is an indication that the spectrophotometer performance may have degraded and that service may be required.
A second context is the measurement of sample transmittance where it is desirable to compensate for the effects of stray light. As described more fully hereinbelow, such compensation has not been heretofore possible to any significant degree of accuracy, and thus stray light has proven to be a source of uncertainty in transmittance measurements.
With respect first to the measurement of stray light as an indication of instrument performance, one accepted method of measuring stray light is set forth in the "Standard Method of Estimating Stray Radiant Energy" published by the American Society for Testing Materials (ASTM) (ANSI/ASTM E387-72 Reapproved 1977). The ASTM method defines the resulting measurement as stray radiant energy (SRE).
Briefly, such a method involves the measurement of the transmittance of a test material at a wavelength where the material is known to be essentially opaque. The observed transmittance is considered to arise solely from stray light and is directly equal to SRE.
As an example of the ASTM method, one accepted test material for use therewith is a one centimeter path length aqueous solution containing 50 g/L of NaNO.sub.2. Such a test solution exhibits a relatively sharp transmittance cutoff at about 400 nm and is essentially opaque in a range of wavelengths from 300 nm to 385 nm (reaching an A of about 16.8 at 355 nm). With the monochromator dial setting adjusted to a wavelength within the 300-385 nm range, the transmittance of the test solution is measured.
For example, with the dial setting at 355 nm and assuming the monochromator half bandwidth to be 2 nm, then primary radiation is radiation having wavelengths between 353 nm and 357 nm. Such primary radiation interval is relatively narrow as compared to the 300-385 nm absorption range of the test solution. Thus, the test solution is highly absorbing throughout the primary radiation interval and essentially absorbs all of the radiation therein. If the monochromator were perfect and the output beam included only primary radiation, essentially no radiation would be transmitted by the test sample and thus no radiant power would be detected by the detector.
However, the monochromator output beam also includes stray light having wavelengths outside the interval of primary radiation. More importantly, a portion of the stray light has wavelengths which are not absorbed by the test solution and therefore pass through the test solution to the detector. Thus, even though the monochromator dial setting is in a wavelength region where the test solution absorbs in essence all of the primary radiation, stray light still reaches the detector. The amount of the detected stray radiant power divided by the total detected radiant power when the test material is not in the beam is called the SRE and gives some indication of instrument performance.
The ASTM method of measuring SRE is deficient in several respects. First, the method can only be performed at wavelengths defined by the spectral characteristics of known and accepted test materials. Such wavelengths may not fully cover the wavelength range of an instrument to be tested. Consequently, instrument performance specifications for stray light using the ASTM method are often given for only one wavelength within the instrument wavelength range, an inadequate indication of instrument performance.
Another important drawback of the ASTM method is that the test material always absorbs at least some of the stray light. In the example described above, the NaNO.sub.2 solution absorbs essentially all radiation within a 300-385 nm range. The extent to which the test material absorbs stray light introduces an error into the measurement of SRE.
Also, the ASTM method of determining SRE is not an accurate measure of stray light as previously defined, which is light having wavelengths outside the interval of primary radiation. In order to perform such an experimental measurement using the ASTM method, it would be necessary to use a test material that is essentially opaque only over the relatively narrow primary radiation interval. Such a test material does not exist.
In an effort to overcome the limitations of the ASTM SRE measurement method, Dr. Kaye, the inventor named herein, developed a stray light measurement technique for determining instrument performance. Dr. Kaye's technique did not require a test material as used in the ASTM method and thus overcame the difficulties associated with test material selection and use. In addition, Dr. Kaye's measurement technique could be automated in a spectrophotometer to provide the user with an indication of current instrument stray light performance for comparison to the original instrument stray light specification. Such automation has been implemented in the model DU.RTM.-5 and model 42 instruments manufactured by Beckman Instruments, Inc., the assignee of the present invention.
In order to present an example of the measurement technique, it is to be noted that the DU-5 and model 42 instruments internally define a plurality of fixed wavelength intervals which limit detector response to radiation with wavelengths within the respective interval. For each monochromator dial setting within the instrument wavelength operating range, one of the fixed wavelength intervals is selected such that the selected fixed wavelength interval includes the monochromator dial setting. The fixed wavelength intervals are defined by the spectral sensitivity characteristics of the source radiation, by the spectral sensitivity characteristics of the detector, by blocking filters or combinations thereof. For example, a fixed wavelength interval can be defined by a bandpass blocking filter placed in the monochromator output beam. Such a blocking filter passes radiation within the fixed wavelength interval but is essentially opaque to radiation with wavelengths outside the fixed wavelength interval. Consequently, radiation with wavelengths outside the fixed wavelength interval is blocked from the detector, thus limiting detector response to radiation with wavelengths within the fixed wavelength interval.
In the DU-5 and model 42 instruments, the operator first selects the stray light test. The instrument then responds with visual instructions to select a test wavelength, .lambda..sub.1, to remove any sample from the beam and to place a reference material or blank into the beam. Once the operator follows the instructions, the operator commands the instrument to automatically continue the performance measurement. In doing so, the monochromator dial setting is adjusted to the test wavelength .lambda..sub.1 and the proper fixed wavelength interval including .lambda..sub.1 is selected. Such selected fixed wavelength interval may be defined by a bandpass blocking filter as previously described. A first detected radiant power (DRP.sub.1) measurement is then made. It is to be noted that with the dial setting adjusted to .lambda..sub.1, the DRP is in response to both primary and stray radiation in the monochromator output beam having wavelengths within the selected fixed wavelength interval.
Once DRP.sub.1 at .lambda..sub.1 is determined, the monochromator dial setting is adjusted to a second wavelength .lambda..sub.2 outside the selected fixed wavelength interval in which DRP.sub.1 was measured. However, the bandpass blocking filter and thus the selected fixed wavelength interval are not changed. Thus, the instrument continues to operate as though the monochromator dial setting is within the selected fixed wavelength interval first selected for .lambda..sub.1. With the monochromator dial setting thus adjusted, a second DRP, DRP.sub.2, is measured. Because .lambda..sub.2 is outside the selected fixed wavelength interval, primary radiation in the monochromator output beam does not effectively contribute to DRP.sub.2. However, the monochromator output beam also includes stray light as previously described. Such stray light having wavelengths within the selected fixed wavelength interval continues to fall on the detector. Hence, DRP.sub.2 is essentially in response to only the stray light within the selected fixed wavelength interval. To find the relative intensity of the stray light at .lambda..sub.1, the ratio DRP.sub.2 /DRP.sub.1 is calculated similar to transmittance and is expressed as a percentage.
Although the technique just described is a substantial improvement over the ASTM standard method because it does not require a separate test material, the resulting instrument performance measurement is based on the assumption that the stray light measured with the dial setting adjusted to .lambda..sub.2 is equal to the stray light present with the dial setting adjusted to .lambda..sub.1. It should be noted that a similar assumption is inherent in the ASTM SRE measurement in that the SRE is assumed to remain constant in the vicinity of the absorption edge of the test material. Such an assumption results in providing an approximation of instrument stray light performance at .lambda..sub.1. Where a more precise measure of stray light is required, such a technique as just described may not provide the needed precision. Thus, there exists a need for an instrument performance stray light measurement method that provides more accurate results.
As noted previously, a second context in which stray light is of interest is in the measurement of sample transmittance (or absorbance). Particularly, stray light can be of interest where a transmittance measurement is made of a sample having a relatively low transmittance (e.g. about 10.sup.-2 or lower) and where the sample transmittance is not constant at all wavelengths.
An example of this can be seen with reference to the above discussion of the ASTM SRE method. The NaNO.sub.2 test solution essentially blocks all radiation having a wavelength of 355 nm. However, the solution does pass radiation having other wavelengths, notably at wavelengths greater than 420 nm. If one were to attempt to measure the transmittance of the test solution of NaNO.sub.2 at, for example, 355 nm, the test solution would absorb essentially all of the light in the monochromator narrow wavelength band centered at 355 nm, that is, essentially all of the primary radiation. However, stray light in the monochromator output beam with wavelengths greater than about 420 nm is transmitted by the solution to the detector, giving rise to a detector output. Because the absorbance of the test solution at 355 nm is approximately 16.8, the resulting transmittance measurement based on the detector output results essentially entirely from stray light passing through the solution at wavelengths where the solution is not highly absorbing. If such a transmittance measurement were not corrected or compensated for stray light, the error inherent therein could have a serious impact on the conclusions drawn from the spectroscopic analysis of the test solution.
In an effort to reduce the influence of stray light on sample measurements, it is known in the art to employ two monochromators in the instrument. Such double monochromator instruments, however, are costly and the two monochromators significantly reduce the intensity of the beam.
It has also been known to attempt to correct transmission data for the effects of stray light. For example, A. Opler in the Journal of the Optical Society of America, Volume 40, page 401 (1950), presented tabulated correction factors for the errors expected from unabsorbed stray light. However, Opler, as well as others, have heretofore failed to accurately account for the influence that the sample will have on stray light. Unless the fraction of stray light absorbed by the sample is known or can be determined, transmission correction is merely speculative and may well under or over correct the transmittance measurement. Moreover, the sign of an error attributable to stray light (i.e., whether the sample transmittance value should be increased or decreased) depends upon whether the sample or the reference absorbs more of the radiation within the monochromator narrow wavelength band or the stray light radiation. Thus, it is possible to increase an error due to stray light if the attempted correction or compensation amount is of the wrong sign.
Heretofore, no method existed for accurately correcting a sample transmittance value for stray light. The ASTM method for the measurement of SRE provides an SRE only for an instrument in the presence of the test material. While such an SRE may be useful in establishing comparisons of SRE between instruments, the SRE provides no accurate predictive value as to the influence that stray light will have on the measured transmittance of other samples. Moreover, because SRE depends upon the presence of the test material in the beam, sample transmittance could not be measured at all in the wavelength region where the test material is essentially opaque. Also, in instruments which define a plurality of fixed wavelength intervals as previously described, the SRE becomes less accurate for a fixed wavelength interval as the interval becomes narrower. Such a result occurs because the stray light within the fixed wavelength interval may be almost completely absorbed by the test material.
The instrument performance measurement technique described above and as used in the model DU-5 and model 42 instruments, provides a more accurate instrument specification. However, even if a sample were inadvertently left in the beam for the automatic measurement performance measurement despite the operator instructions to the contrary, the resulting ratio would be meaningless and of no use in a stray light compensation context.
Thus, there is a need for a method for compensating transmittance and absorbance measurements for stray light.